An extracellular protease of Acremonium chrysogenum with CPC-acetylhydrolase activity and its use for the synthesis of deacetylated derivatives of cephalosporin C and inactivation of the gene for increasing the yield of cephalosporin.
The present invention includes the identification of a protein with CPC-acetylhydrolase (CPC-AH) activity secreted by the fungus Acremonium chrysogenum, purification of the said protein, cloning of the gene that codes for the said enzyme and the inactivation of this gene by genetic methods. In addition it proposes use of the enzyme for the preparation of deacetylated derivatives of cephalosporin C and 7-aminocephalosporanic acid (7-ACA), such as deacetylcephalosporin C (DAC) and deacetyl 7-ACA. These compounds can be used as starting material for the synthesis of some derivatives of cephalosporin of commercial interest.
In industrial fermentations of A. chrysogenum for the production of cephalosporin, accumulation of DAC in the culture media is observed. This accumulation is due on the one hand to insufficient acetylation of the DAC and on the other hand to chemical and enzymatic deacetylation of the CPC formed. Inactivation by using recombinant DNA techniques of the gene cahB, which codes for the enzyme CPC-AH B, partly prevents accumulation of DAC during fermentation.
The production of deacetylcephalosporin C (called DAC hereinafter) from cephalosporin C (called CPC hereinafter) is effected by eliminating the acetyl residue at the 3xe2x80x2 carbon of the CPC molecule. This conversion can be carried out by chemical or enzymatic methods. Advantages of the latter are that they can be effected in less drastic conditions of pH and temperature and in addition they produce fewer side reactions. The use of enzymes with cephalosporin C-acetylhydrolase (hereinafter referred to as CPC-AH) activity obtained from a variety of sources such as the peel of citrus fruits (Jeffery et al., 1961, Biochem. J. 81: 591-596), actinomycetes (Demain et al., 1963, J. Bacteriol. 35: 339-344), Bacillus subtilis (Abbott and Fukuda, 1975, Appl. Microbiol. 30: 413-419; Takimoto et al., 1994, J. Ferment. Bioeng. 77: 17-22), Rodosporidium toruloides (Politino et al., 1997, Appl. Env. Microbiol. 63: 4807-4811) and Thermoanaerobium sp. (Lorenz and Wiegel, 1997, J. Bacteriol. 179: 5436-5441), is known.
On the other hand, appreciable amounts of DAC have been detected both in intracellular extracts and in extracellular fluids of A. chrysogenum, the fungus used for the industrial production of CPC. The presence of this CPC precursor is undesirable because it lowers the final yields of CPC and hampers its purification. Various non-exclusive hypotheses have been proposed to explain the origin of this compound in the culture media: (I) Some of the DAC accumulated may originate from lack of acetylation of the biosynthetic intermediate, as it is known that there is a low level of expression of the cefG gene that codes for DAC-acetyltransferase (Velasco et al., 1994, J. Bacteriol. 176: 985-991). (II) The DAC may arise through chemical hydrolysis of CPC which is more evident at alkaline pH (Konecny et al., 1972, J. Antibiot. 26: 135-141). (III) The DAC may be synthesized by enzymatic hydrolysis, as extracellular CPC-AH activities have been described in fermentation media of A. chrysogenum. Fujisawa et al. (1975, Agric. Biol. Chem., 39: 1303-1309) described a mutant with CPC-AH activity. Shortly thereafter, Hinnen and Nxc3xcesch (1976, Antimicrob. Agents Chemother. 9: 824-830) purified an extracellular CPC-AH with low affinity for CPC (kM 20 mM) and whose synthesis is controlled by the carbon source. The purified CPC-AH had a molecular weight of approximately 25 kDa and an isoelectric point of 4.3. The enzyme was strongly inhibited by diisopropylfluorophosphate. However, these preliminary studies have not been followed by any description of more detailed data concerning the said activities, nor the cloning, characterization and use of the corresponding gene.
In accordance with the invention, there is provided an isolated DNA sequence comprising SEQ ID NO:4 or a fragment of SEQ ID NO:4 encoding a peptide with CPC-acetylhydrolase enzymatic activity.
There is also provided a method of expressing CPC-acetylhydrolase activity comprising:
(a) providing a microorganism that is susceptible to transformation with the isolated DNA sequence and that, upon transformation, expresses the CPC-acetylhydrolase activity encoded by said DNA sequence; and
(b) transforming the microorganism with the DNA sequence to cause expression of the CPC-acetylhydrolase activity in the microorganism. In one embodiment, the method further comprises using the expressed CPC-acetylhydrolase activity to prepare a deacetylated derivative of cephalosporin C or 7-aminocephalosporanic acid. The derivative can be deacetylcephalosporin C or deacetyl 7-aminocephalosporanic acid.
There is further provided a method for producing a microorganism with increased capacity to aid in the production of cephalosporin comprising
(a) providing a microorganism that has CPC-AH activity by virtue of expression of the DNA sequence or a fragment thereof coding for the CPC-acetylhydrolase enzymatic activity; and
(b) inactivating said activity by disrupting expression of the DNA sequence.